Hemophilia B (also known as Christmas disease) is one of the most common inherited bleeding disorders in the world. It results in decreased in vivo and in vitro blood clotting activity and requires extensive medical monitoring throughout the life of the affected individual. In the absence of intervention, the afflicted individual may suffer from spontaneous bleeding in the joints, which produces severe pain and debilitating immobility; bleeding into muscles results in the accumulation of blood in those tissues; spontaneous bleeding in the throat and neck may cause asphyxiation if not immediately treated; bleeding into the urine; and severe bleeding following surgery, minor accidental injuries, or dental extractions also are prevalent.
To the extent that the present invention relates to intervention of blood clotting disorders, a brief discussion of the biological factors and/or mechanisms involved in blood clotting is warranted. A blood clot is essentially a gelatinous mass, which seals blood vessels that have sustained an injury. Conversion of fluid blood to a blood clot involves the conversion of soluble fibrinogen, which is present in plasma, to the insoluble gelatinous blood clot, composed primarily of cross-linked fibrin. The conversion of fibrinogen to fibrin is the primary end result of a multi-step process referred to as the blood coagulation cascade. This-cascade is a highly regulated process that involves the sequential proteolytic conversion of serine proteases from zymogen to active conformations, and subsequent formation of calcium-dependent phospholipid-bound enzyme complexes with specific protein cofactors. Normal in vivo blood coagulation at minimum requires the serine proteases factors II (prothrombin), VII, IX, X and XI (soluble plasma proteins); cofactors including the transmembrane protein tissue factor and the plasma proteins factors V and VIII; fibrinogen, the transglutaminase factor XIII, phospholipid (including activated platelets), and calcium. Additional proteins including kallikrein, high molecular weight kininogen, and factor XII are required for some in vitro clotting tests, and may play a role in vivo under pathologic conditions. The coagulation cascade is regulated by the thrombomodulin-protein C pathway, the fibrinolysis pathway, tissue factor pathway inhibitor, and the serpin antithrombin III. Importantly, the inhibition of several coagulation proteases by antithrombin III (including factor IXa) is markedly accelerated by the anticoagulant drug heparin, as well as structurally similar heparan sulfate on the endothelial surface.
Upon injury, thrombocytes, in the presence of von Willebrand Factor (a component of clotting Factor VIII), cling to the collagen of injured connective tissue by adhesion. The thrombocytes change their form and develop protrusions, and in addition to this, their outer membrane facilitates the adhesion of further thrombocytes. Thereafter, various substances are released from granula of these cells, which results in vessel constriction as well as accumulation and activation of other factors of plasma blood clotting.
In hemophilia, blood clotting is disturbed by a lack of certain plasma blood clotting factors. Hemophilia B is caused by a deficiency in factor IX that may result from either the decreased synthesis of the factor IX protein or a defective molecule with reduced activity. The treatment of hemophilia occurs by replacement of the missing clotting factor by exogenous factor concentrates highly enriched in factor IX. However, generating such a concentrate is fraught with technical difficulties as described below.
Factor IX, like other clotting factors, is naturally produced as a precursor molecule having an additional pre-pro-sequence at the N-terminus. The pre-pro-sequence represents a signal sequence that causes the oriented transport of this protein in the cell. When the pre-pro Factor DC protein is secreted from the cell the pre-sequence is cleaved. The pro-sequence consists of about 15 to 18 amino acids and serves as a recognition sequence in carboxylation of glutamic acid residues to 4-carboxy-L-glutamic acid. After successful carboxylation, the pro-sequence is also cleaved. If the pro-sequence is not cleaved or only incompletely cleaved, only low activity clotting factors result. Human factor IX has a molecular weight of about 55,000 Dalton; when its pro-sequence is present the molecular weight is increased by about 2000 Dalton.
Purification of factor IX from plasma almost exclusively yields active factor IX. However, such purification of factor IX from plasma is very difficult because factor IX is only present in low concentration in plasma [5 μg/mL; Andersson, Thrombosis Research 7: 451–459 (1975)]. Efforts to produce recombinant factor IX have led to products with only low levels of activity [Kaufman et al., J. Biol Chem 261: 9622–9628 (1986); Busby et al., Nature 316: 217–273 (1985); Rees et al., EMBO J 7: 2053–2061 (1988)]. This can be traced back to an incomplete cleavage of the pro-sequence [Meulien et al., Prot Engineer 3: 629–633 (1990)] because a mixture of recombinantly produced pro-factor IX and factor IX is present in cell supernatants.
The in vivo activity of exogenously generated factor IX is limited both by protein half-life and inhibitors of coagulation, including antithrombin III. An additional factor that limits the use of exogenously generated factor IX in an effective therapeutic protocol is that endogenous heparan sulfate/heparin greatly inhibits the activity of factor IX that is used in the existing therapies for hemophilia B.
Heparin can inhibit factor IXa activity in the intrinsic tenase complex (factor IXa-factor VIIIa) directly, or markedly accelerate inhibition of factor IXa by antithrombin III. Heparan sulfate, a chemically similar glycosaminoglycan to heparin, is expressed widely in the body including the endothelial surface, where it has been demonstrated to accelerate inhibition of coagulation proteases by antithrombin III. Similarly, it may directly inhibit intrinsic tenase activity at sites of injury, limiting the in vivo activity of factor IXa. Thus, a mutant factor IXa that is resistant to the effects of heparin/heparan sulfate and retains in vitro clotting activity may have enhanced in vivo activity. Similar protein engineering approaches have been used to enhance the therapeutic efficacy of other serine proteases, including improvement of the fibrin binding properties of tissue plasminogen activator by mutagenesis.
Thus, there is a need for mutant factor IX, which has a reduced affinity for heparin but retains it anti-clotting activity, and remains active when administered as part of a therapeutic regimen for hemophilia B.